Determining the genomic location and the chromosomal flanking sequence adjacent to an inserted transgene is technically challenging. Various methods have been developed to overcome the limitation of identifying the unknown DNA sequences which flank a known DNA sequence. However, these traditional PCR methods for the identification or genomic chromosomal sequences which flank a known transgene such as LM-PCR (also described as Genome Walking) and other methods including: inverse PCR (i-PCR), thermal asymmetric interlaced PCR (TAIL-PCR), anchored PCR (a-PCR) and randomly primed PCR (rm-PCR) are hindered by low detection sensitivity (requiring large quantities of template DNA) or low specificity because of losses of DNA during preparation.
The polymerase chain reaction (PCR) is a commonly employed molecular biology method. The method is performed by denaturing double-stranded template DNA, annealing oligonucleotide primers to the DNA template, and extension of a DNA strand via a DNA polymerase. The oligonucleotide primers are designed to anneal to opposite strands of the DNA and positioned so that the DNA strand produced by the DNA polymerase serves as a template strand for the other primer. Each cycle is repeated, resulting in the exponential amplification of a DNA fragment. (Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). The use of PCR by those skilled in the art is fundamental for amplifying and isolating DNA fragments for subsequent analysis.
Isolation and analysis of DNA templates via the polymerase chain reaction (PCR) requires knowledge of the flanking DNA sequences. Unfortunately, this requirement limits PCR amplification to regions of known DNA sequence. The use of PCR methodologies to identify the location of a transgene location within a genome is hindered by the random insertion of the transgene into an unknown chromosomal location within the genome of an organism. Methods to identify unknown DNA sequences which are located adjacent to a known DNA sequence are necessary for the identification of a transgene location within the chromosome of an organism. In addition such methods can be used to identify novel gene sequences to identify new traits, to determine the genomic location of a transposon or viral sequence which has been inserted into the genome of an organism, or to identify the chromosomal location of polynucleotide sequences inserted into the genome via insertion mutagenesis.
Various methods have been developed to overcome the limitation of the unknown DNA sequences which flank a known DNA sequence. A Ligation Mediated PCR (LM PCR) method wherein a genomic library is generated and adapters are annealed to DNA fragments for PCR amplification is marketed as the GENOME WALKER UNIVERSAL KIT™ (see U.S. Pat. No. 5,565,340, and U.S. Pat. No. 5,759,822). Another method commonly used is the inverse PCR reaction (see Silver and Keerikatte (1989), J. Virol., 63:1924-1928), wherein DNA is digested with a restriction enzyme and self ligated resulting in a contiguous circle. PCR amplification using oligonucleotide primers which bind to known sequences results in amplification and elucidation of the unknown flanking sequences. Unfortunately these methods are inefficient and time consuming. These and other traditional PCR methods (including thermal asymmetric interlaced PCR [TAIL-PCR], anchored PCR [a-PCR] and randomly primed PCR [rm-PCR]) are hindered by low detection sensitivity (requiring large quantities of template DNA) or low specificity because of losses of DNA during preparation.
The development of a method which can improve detection sensitivity by purifying chromosomal DNA fragments which contain both the known and unknown DNA sequences can result in a sensitive method for detecting and characterizing unknown DNA regions which are located adjacent to a known DNA sequence. The development of the Linear Amplification Mediated Polymerase Chain Reaction (LAM PCR) method achieves these goals. see U.S. Pat. No. 6,514,706. The LAM PCR method is particularly suited to amplify and analyze DNA fragments, the sequence of which is only known in part.
The development of a method which can improve detection sensitivity by purifying chromosomal DNA fragments which contain both the known and unknown DNA sequences can result in a sensitive method for detecting and characterizing unknown DNA regions which are located adjacent to a known DNA sequence. The development of the LAM PCR method achieves these goals. LAM PCR is a modified PCR method that is used for analyzing unknown chromosomal flanking sequences located adjacent to a known DNA sequence. The LAM PCR method can be used to identify and/or sequence an unknown DNA or RNA sequence flanking a known DNA or RNA region.
The LAM PCR method consists of the following steps. A primer extension reaction is performed using a chromosomal DNA as a template and an oligonucleotide primer which binds to a known DNA sequence within the chromosomal DNA. The oligonucleotide primer is complementary to a long terminal repeat (LTR) sequence, which is a sequence characteristic of a retrovirus, and labeled with biotin at the end of the oligonucleotide primer. The single-stranded DNA product of the linear PCR is bound to magnetic beads having immobilized streptavidin. This step serves to isolate the single-stranded amplified DNA fragment containing the known LTR sequence and an unknown sequence derived from the chromosome. The single-stranded DNA is converted into a double-stranded DNA by synthesizing the complementary strand. The double-stranded DNA is cleaved with a restriction enzyme that recognizes a sequence and cleaves the double-stranded DNA at the sequence. A double-stranded DNA called a linker cassette is ligated to the terminus. Subsequent PCR reactions are conducted using the thus obtained ligation product as a template as well as a primer complementary to the LTR and a primer complementary to the linker cassette. A DNA fragment that contains the LTR and chromosome DNA flanking sequence adjacent to the LTR is amplified. As a result the previously unknown retrovirus integration site can be determined.
The LAM PCR method is currently considered to be an effective system for analyzing unknown DNA sequences adjacent to a known DNA sequence. Modifications and improvements to the LAM PCR method have been described in the art. see U.S. Pat. App. US2007/0037139 and Harkey et al., (2007) Stem Cells Dev., June; 16(3): 381-392.
The LAM PCR method was modified in U.S. Pat. App. US2007/0037139 to improve the detection of a biological sample having a retrovirus integrated at various sites. The reaction conditions of the traditional LAM PCR method produced results that did not reflect the actual state of clones existing in the cell population of the sample. A modification was developed in which more integration fragments were PCR amplified without being biased toward a fragment amplified from a specific clone. The modification to the LAM PCR method allowed researchers to determine the extent of cells having an integrated gene in the population and to determine the ratio of a specific cell in the population.
In addition, Harkey et al., (2007) describe an optimized, multi-arm, high throughput modification of the LAM PCR method wherein the detection capacity was improved 90% with exhaustive sampling. The modified protocol facilitated accurate estimates of the total pool size, thus providing a rapid, cost-effective approach for generating large insertion-site data of preferred genomic locations for vector integration.
The subject invention describes a further significant modification and solves several traditional LAM-PCR problems by eliminating the steps of generating a double stranded DNA fragment then digesting the double stranded DNA fragment and denaturing the double stranded DNA fragment.